Hepatic insulin resistance due to obesity is central to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). This research proposal addresses the unanswered question of how molecular mechanisms that normally promote energy conservation contribute to NAFLD in obese individuals. The long-term goal of this research is to understand the regulatory relationships between cellular lipid molecules and metabolism, particularly as they present therapeutic opportunities. The objective of this research is to understand fundamental new mechanisms for the regulation of energy homeostasis and nutrient metabolism. The central hypothesis is that thioesterase superfamily member 1 (Them1) functions in brown adipose tissue (BAT) as a lipid-regulated fatty acyl-CoA thioesterase that controls intracellular fatty acid trafficking and that regulates the expression of thermogenic genes. In the setting of obesity, we postulate that upregulation of Them1 in liver and white adipose tissue promotes endoplasmic reticulum (ER) stress, mitochondrial dysfunction and inflammation due to the overproduction of free fatty acids. The rationale is that the mechanisms of Them1-mediated metabolic regulation should yield new insights into the pathogenesis of NAFLD. Guided by extensive preliminary data, the central hypothesis will be tested in three specific aims: 1) To determine the mechanisms whereby Them1 limits energy expenditure in BAT; 2) To demonstrate a primary pathogenic role for Them1 in NAFLD; and 3) To elucidate molecular regulation of Them1 activity by the lipid-binding steroidogenic acute regulatory protein-related lipid transfer (START) domain. In Aim 1, recently developed Them1-/- mice and cultured brown adipocytes will be used to test the hypothesis that Them1 in BAT limits access of fatty acids to mitochondria and reduces the expression of thermogenic genes. Aim 2 will leverage newly created transgenic mice, as well as cell culture systems to explore whether Them1 contributes directly to hepatic steatosis and insulin resistance. Mice with liver-specific overexpression will be used to establish the role of Them1 in promoting hepatic ER stress, mitochondrial dysfunction and inflammation. Mice with adipose-specific overexpression will reveal the contributions of Them1 to inflammation within white adipose tissue and to hepatic steatosis. Aim 3 will determine the lipid ligand(s) of the Them1 START domain by mass spectrometry. X-ray crystallography will be used to determine the structure of Them1 in complex with its ligands, the relationship among functional domains and the influence of lipid binding on enzymatic activity. High throughput screening will identity small molecule inhibitors, which should facilitate structure-function studies and help delineate the biological roles of Them1. Overall, this proposal will elucidate Them1-mediated metabolic regulation, which is significant because mechanisms that conserve energy in health may promote disease under conditions of overnutrition. These studies are expected to identify new therapeutic targets for the management of NAFLD.